The study introduces a novel proteomics method, isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis – Activity-Based Protein Profiling), to quantitatively profile the intrinsic reactivity of cysteine residues in native biological systems. This method uses an electrophilic iodoacetamide (IA) probe, which is labeled to cysteine residues in proteins, followed by click chemistry conjugation and isotopic labeling. The method allows for the quantitative analysis of cysteine reactivity by comparing the intensity of light and heavy tag-conjugated proteomes. The authors found that hyperreactive cysteines, which are rare, are associated with a wide range of activities, including nucleophilic and reductive catalysis, and sites of oxidative modification. Hyperreactive cysteines were identified in uncharacterized proteins, including a conserved residue in yeast that is essential for viability and involved in iron-sulfur protein biogenesis. The method also demonstrated the ability to screen and assign functional roles to cysteines in computationally designed proteins, where it distinguished catalytically active from inactive cysteine hydrolase designs. The study highlights the importance of cysteine reactivity in protein function and provides a robust approach to characterizing cysteine functionality on a global scale.The study introduces a novel proteomics method, isoTOP-ABPP (isotopic Tandem Orthogonal Proteolysis – Activity-Based Protein Profiling), to quantitatively profile the intrinsic reactivity of cysteine residues in native biological systems. This method uses an electrophilic iodoacetamide (IA) probe, which is labeled to cysteine residues in proteins, followed by click chemistry conjugation and isotopic labeling. The method allows for the quantitative analysis of cysteine reactivity by comparing the intensity of light and heavy tag-conjugated proteomes. The authors found that hyperreactive cysteines, which are rare, are associated with a wide range of activities, including nucleophilic and reductive catalysis, and sites of oxidative modification. Hyperreactive cysteines were identified in uncharacterized proteins, including a conserved residue in yeast that is essential for viability and involved in iron-sulfur protein biogenesis. The method also demonstrated the ability to screen and assign functional roles to cysteines in computationally designed proteins, where it distinguished catalytically active from inactive cysteine hydrolase designs. The study highlights the importance of cysteine reactivity in protein function and provides a robust approach to characterizing cysteine functionality on a global scale.